Peripheral artery disease (PAD) is a common condition in which blood flow is reduced to the limbs, typically the leg and feet (Manzi et al., 2011; Stansby and Williams, 2011), and if untreated, may progress to the stage of critical limb ischemia (CLI), which is the most advanced form of PAD often leading to amputation of the limb and potential mortality (Chan and Cheng, 2011; Dattilo and Casserly, 2011). Similar to myocardial infarction (MI), which is also a result of atherosclerosis, PAD has a large population of affected individuals, with an estimated 27 million suffering from PAD in Europe and North America (Belch et al., 2003). Despite recent medical advances and the advent of tissue engineering strategies, revascularization through surgical or endovascular intervention, remains the only treatment. This is further complicated by the fact that approximately 40% of patients with critical limb ischemia (CLI) are not candidates for revascularization procedures (Sprengers et al., 2008), and that revascularization has limited benefit when the PAD is diffuse or below the knee. This corresponds to approximately 120,000 leg amputations in the US and 100,000 in the European Union each year (Lawall et al., 2010). There is therefore a pressing need for the development of new therapies for treating PAD and CLI.
Alternative therapies for PAD and CLI have largely mirrored the attempts for MI and heart failure, including cell transplantation and angiogenic growth factor therapy (Fadini et al., 2010; Menasché, 2010; Tongers et al., 2008). The goal of these therapies has been to increase vascularization of the ischemic limb so that perfusion is sufficient for wound healing to occur, and to resolve pain at rest, thereby also preventing limb amputations. Biomaterial based strategies have recently been explored. Currently, only poly(d,1-lactide-co-glycolide) (PLGA), collagen-fibronectin, alginate, gelatin, fibrin, and peptide amphiphiles have been examined (Doi et al., 2007; Jay et al., 2008; Kong et al., 2008; Layman et al., 2007; Lee et al., 2010; Ruvinov et al., 2010; Silva and Mooney, 2007; Webber et al., 2011). PLGA microspheres in alginate hydrogels have been utilized to deliver vascular endothelial growth factor (VEGF) (Lee et al., 2010), and alginate hydrogels have been explored for delivery of hepatocyte growth factor (HGF) (Ruvinov et al., 2010), VEGF (Silva and Mooney, 2007), and pDNA encoding for VEGF (Kong et al., 2008). Alginate microspheres within an injectable collagen matrix have also been used to deliver stromal cell-derived factor-1 (SDF-1) (Kuraitis et al., 2011), while VEGF loaded alginate microparticles in a collagen-fibronectin scaffold were used to deliver endothelial cells (Jay et al., 2008). Gelatin hydrogels have been employed to deliver basic fibroblast growth factor (bFGF) (Doi et al., 2007; Layman et al., 2007), and fibrin scaffolds were utilized to deliver bFGF and granulocyte-colony stimulating factor (G-CSF) along with bone marrow cells. More recently, VEGF-mimetic peptides were delivered using assembling peptide amphiphiles (Webber et al., 2011). Each of these studies demonstrated enhanced cell transplantation and/or enhanced growth factor/gene delivery with resulting enhancements in neovascularization.
While each of the materials currently explored for treating PAD have been used extensively as tissue engineering scaffolds, they do not mimic the extracellular microenvironment of the skeletal muscle they are intended to treat.
Recently, several clinical trials using cell therapy have demonstrated promising results (Alev et al., 2011; Gupta and Losordo, 2011; Kawamoto et al., 2009; Menasché, 2010), but there are still many questions about which therapeutic cell type to use, quantity of cells, and best route to deliver the cells, as well as a significant problem with poor cell retention and survival (Menasché, 2010). Biomaterial scaffolds have more recently been explored to enhance cell survival by providing a temporary mimic of the extracellular matrix (ECM) (Kawamoto et al., 2009; Layman et al., 2011). Biomaterials have also been used for delivery of growth factors or their mimics in animal models of PAD and CLI (Kawamoto et al., 2009; Layman et al., 2011; Ruvinov et al., 2010; Webber et al., 2011). However, these scaffolds are composed of fibrin (Layman et al., 2011), collagen-based matrix (Kuraitis et al., 2011), gelatin (Layman et al., 2007), self-assembling peptide amphiphiles (Webber et al., 2011) or alginate (Ruvinov et al., 2010), which may not provide the proper biomimetic environment for the ischemic skeletal muscle in these conditions. Moreover, no potential therapies employing only a biomaterial have been explored to date. An acellular, biomaterial only approach may reach the clinic sooner since it has the potential to be an off-the-shelf treatment and does not have the added complications that cells bring, including appropriate source, the need for expansion, or potential disease transmission. Furthermore, a biomaterial that promotes neovascularization and tissue repair on its own would obviate the need for exogenous growth factors, and the difficulties and expense associated with such a combination product.
The extracellular matrix (ECM) of each tissue contains similar components; however, each individual tissue is composed of a unique combination of proteins and proteoglycans (Lutolf and Hubbell, 2005; Uriel et al., 2009). Recent studies have shown that the ECM of various tissues can be isolated through decellularization and utilized as a tissue engineering scaffold (Merritt et al.; Ott et al., 2008; Singelyn et al., 2009; Uygun et al., 2010; Valentin et al., 2010; Young et al., 2011). Other decellularized ECM materials have been used for a variety of applications for tissue repair (Crapo et al., 2011; Gilbert et al., 2006). These scaffolds are known to promote cellular influx in a variety of tissues (Numata et al., 2004; Rieder et al., 2006). Their degradation products have angiogenic (Li et al., 2004) and chemoattractant (Badylak et al., 2001; Beattie et al., 2008; Li et al., 2004; Zantop et al., 2006) properties, and also promote cell migration and proliferation (Reing et al., 2009). After removal of the cellular antigens, these scaffolds are considered biocompatible, and both allogeneic and xenogeneic ECM devices have been approved by the FDA and are in clinical use (Badylak, 2007).
Hydrogels derived from decellularized ECMs, including myocardium (Singelyn et al., 2009), pericardium (Seif-Naraghi et al., 2010), and adipose tissue (Young et al., 2011), were recently developed which assemble into porous and fibrous scaffolds upon injection in vivo. It is also recently shown that the injectable hydrogel derived from ventricular ECM promoted endogenous cardiomyocyte survival and preserved cardiac function post-myocardial infarction (Singelyn et al., 2012).
Skeletal muscles are composed of bundles of highly oriented and dense muscle fibers, each a multinucleated cell derived from myoblasts. The muscle fibers in native skeletal muscle are closely packed together in an extracellular three-dimensional matrix to form an organized tissue with high cell density and cellular orientation to generate longitudinal contraction. Skeletal muscle can develop scar tissue after injury which leads to a loss of functionality. The engineering of muscle tissue in vitro holds promise for the treatment of skeletal muscle defects as an alternative to host muscle transfer. Tissue engineering compositions must be biocompatible and capable of being vascularised and innervated.
The reconstruction of skeletal muscle, which is lost by injury, tumor resection, or various myopathies, is limited by the lack of functional substitutes. Surgical treatments, such as muscle transplantation and transposition techniques, have had some success; however, there still exists a need for alternative therapies. Tissue engineering approaches offer potential new solutions; however, current options offer incomplete regeneration. Many naturally derived as well as synthetic materials have been explored as scaffolds for skeletal tissue engineering, but none offer a complex mimic of the native skeletal extracellular matrix, which possesses important cues for cell survival, differentiation, and migration.
The extracellular matrix consists of a complex tissue-specific network of proteins and polysaccharides, which help regulate cell growth, survival and differentiation. Despite the complex nature of native ECM, in vitro cell studies traditionally assess cell behavior on single ECM component coatings, thus posing limitations on translating findings from in vitro cell studies to the in vivo setting. Overcoming this limitation is important for cell-mediated therapies, which rely on cultured and expanded cells retaining native cell behavior over time.
Typically, purified matrix proteins from various animal sources are adsorbed to cell culture substrates to provide a protein substrate for cell attachment and to modify cellular behavior. However, these approaches would not provide an accurate representation of the complex microenvironment. More complex coatings have been used, such as a combination of single proteins, and while these combinatorial signals have shown to affect cell behavior, it is not as complete as in vivo. For a more natural matrix, cell-derived matrices have been used. Matrigel is a complex system; however, it is derived from mouse sarcoma, and does not mimic any natural tissue. While many components of ECM are similar, each tissue or organ has a unique composition, and a tissue specific naturally derived source may prove to be a better mimic of the cell microenvironment.
A liquid form of skeletal muscle matrix was shown to promote the differentiation and maturation of C2C12 skeletal myoblast progenitors when used as a cell culture coating due to its ability to retain a complex mixture of skeletal muscle ECM proteins, peptides, and proteoglycans (DeQuach et al., 2010). A decellularized skeletal muscle scaffold has been previously explored for replacement of a muscle defect (Merritt et al.; Wolf et al., 2012), yet this intact scaffold would not be amenable to treating certain non-skeletal muscle tissue disease, such as the peripheral artery disease (PAD) and CLI.
As discussed above, the only current clinical treatment for PAD and CLI is endovascular or surgical revascularization (Dattilo and Casserly, 2011). Surgical bypass was the established standard, but recently endovascular therapies such as angioplasty, atherectomy and stenting are used as less-invasive options. However, despite these therapies, CLI continues to carry a major risk of limb amputation, with rates that have not changed significantly in 30 years (Tongers et al., 2008). Unfortunately, few therapies exist for treating the ischemic skeletal muscle in these conditions. Biomaterials have been used to increase cell transplant survival as well as deliver growth factors to treat limb ischemia; however, existing materials do not mimic the native skeletal muscle microenvironment they are intended to treat. Furthermore, no therapies involving biomaterials alone have been examined.